mycoplasma false positive

We use the Lonza MycoAlert test kit which works on luminescence to test for mycoplasma in our lab. We recently had 2 cultures that looked contaminated and we took some of the supernatant and plated it onto Agar plates and also placed it into TSB to grow overnight. The plates and TSB cultures looked like a standard bacterial contamination the next morning. We took some of the supernatant of the media and tested it for mycoplasma using the Lonza bioluminescence kit. It has come back as being positive for mycoplasma. The media was frozen before doing the myco testing. My question is this, can some other bugs give false positive for mycoplasma when using the luminescence kits?
We have had bacterial contamination from time to time but have never had a mycoplasma infection before so this is very worrying.

It is a little hard to tell, but I would say that there is a good chance that you have a normal bacterial infection rather than mycoplasma. Try growing some of the cells on a coverslip and staining with a DNA dye (Hoescht 33258 is the usual one) and then looking at the cells under a fluorescent microscope. IF you have mycoplasma there should be lots of tiny spots of DNA outside the nuclei of the cells. Typically these will not be seen in the area outside of the cells.

Most mycoplasma detection kits of the sort you are using are not specific enough to tell you if you have mycoplasma!

Thanks for that! We don't actually have any of the cells in culture any more, we threw them out as soon as we suspected contamination. We're going to send away some of the left over supernatant to an external testing body but you've certainly eased my mind a little.